CAR T cell-based therapies have shown promising results in hematological malignancies. Results of CAR T cell projects in solid tumors have been less impressive, and factors including lack of targetable antigens and immunosuppressive tumor microenvironment (TME) have been suggested as culprits. Adenosine, a metabolite which is highly produced in TME, is known to mediate the suppression of anti-tumor T cell responses via binding and signaling through adenosine 2a receptor (A2aR). In this study, the expression of A2aR and the effects of its activation on the function of fully human anti-mesothelin CAR T cells (MSLN-CAR T), were analyzed. Afterwards, the molecular and pharmacological means to overcome the inhibitory effects of A2aR signaling on CAR T cell function were used. This was performed by targeting A2aR expression in MSLN-CAR T cells using various anti-A2aR shRNA sequences embedded in the CAR vector and an A2aR pharmacological antagonist, SCH-58261. Statistical analyses were performed Prism 7 software. Our experiments showed significant A2aR upregulation on T cells during the CAR T cell production procedure (cell activation and transduction). Activation of adenosine signaling using adenosine analog led to the suppression of all major anti-tumor functions in MSLN-CAR T cells. Interestingly, CAR T cells that carried the anti-A2aR shRNA sequences were resistant to the inhibitory effects of adenosine signaling. Pharmacological inhibition of A2aR reversed the reduction in CAR T cell proliferation and cytokine response caused by the adenosine analog; however, it failed to rescue the cytotoxic function of the cells. Altogether, our results indicate that mitigating A2aR signaling by genetic targeting of the receptor might be a promising approach in improving CAR T cell function in an unreceptive microenvironment and could potentially improve the outcome of treatment in clinical settings.

中文翻译：

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A2a受体的遗传和药理靶向作用可改善抗间皮素CAR T细胞的功能

基于CAR T细胞的疗法已在血液系统恶性肿瘤中显示出令人鼓舞的结果。实体瘤中CAR T细胞项目的结果令人印象深刻，并且已建议罪魁祸首包括缺乏可靶向抗原和免疫抑制性肿瘤微环境（TME）的因素。腺苷是在TME中高产的一种代谢产物，已知通过腺苷2a受体（A2aR）的结合和信号传导来介导抗肿瘤T细胞反应的抑制。在这项研究中，分析了A2aR的表达及其激活对全人类抗间皮素CAR T细胞（MSLN-CAR T）功能的影响。之后，使用克服A2aR信号转导对CAR T细胞功能的抑制作用的分子和药理学手段。这是通过使用嵌入在CAR载体中的各种抗A2aR shRNA序列和A2aR药理拮抗剂SCH-58261靶向MSLN-CAR T细胞中A2aR表达来实现的。进行统计分析，使用Prism 7软件。我们的实验表明，在CAR T细胞产生过程（细胞激活和转导）过程中，T细胞上的A2aR明显上调。使用腺苷类似物激活腺苷信号传导导致抑制MSLN-CAR T细胞中所有主要的抗肿瘤功能。有趣的是，携带抗A2aR shRNA序列的CAR T细胞对腺苷信号转导的抑制作用有抗性。A2aR的药理抑制作用逆转了腺苷类似物引起的CAR T细胞增殖和细胞因子反应的降低；然而，它未能挽救细胞的细胞毒性功能。